애기장대의 개화시기 조절에 중요한 FKF1 LOV 도메인의 아미노산 특성 규명
Characterization of an amino acid in the FKF1 LOV domain that is crucial for the regulation of flowering time in Arabidopsis thaliana
- 주제(키워드) FLAVIN-BINDING , KELCH REPEAT , F-BOX1 (FKF1) , FKF1 I160R , photoperiodic flowering
- 발행기관 아주대학교
- 지도교수 송영훈
- 발행년도 2018
- 학위수여년월 2018. 8
- 학위명 석사
- 학과 및 전공 일반대학원 생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000028119
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Plants keep measuring changes in day length to maximize reproductive fitness under favorable seasons. In Arabidopsis thaliana, a model plant that flowers early in long days, the induction of FLOWERING LOCUS T (FT) gene expression determines the timing of flowering. The FT expression is directly activated by the transcription factor CONSTANS (CO). The diurnal expression patterns of CO gene and protein are crucial for the day length-dependent FT expression. FLAVIN BINDING, KELCH REPEAT, F-BOX1 (FKF1) is a blue light photoreceptor that acts as a photoperiod sensor. FKF1 positively regulates CO gene expression and CO protein stability directly as well as FT gene expression directly and indirectly, which facilitates plants to flower at the right season. The LOV (Light, Oxigen, Voltage) domain in FKF1 is responsible for blue light absorption and plays important roles in the regulation of CO and FT genes by the formation of the protein complex with GIGENTEA (GI) and in the stabilization of CO by the interaction with it. Recent analysis of the LOV domain structure of ZTL, a FKF1 homologue, predicted that the amino acid isoleucine at position 151 plays an important role in the formation of dimerization, which is essential for the unique function of the protein. In FKF1, the 160th amino acid isoleucine has the same structural feature. In this study, I investigated the functional significance of the FKF1 LOV domain in flowering regulation through the substitution of arginine for isoleucine (I160R). The LOV domain mutant form of FKF1 protein (FKF1 I160R) promoted flowering time, although the protein abundance of the form was lower than that of wild type FKF1. Moreover, FKF1 I160R specifically increased FT mRNA levels but not CO mRNA levels. Since no significant differences in the interactions of FKF1 I160R with GI and CO in vivo and in planta, respectively, compared to wild type FKF1, these observations suggested that FKF1 I160R might regulate CO at the post-translational level. Co-immunoprecipitation assays showed an increased binding of FKF1 I160R to CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), which mediates the degradation of CO, in vivo. In conclusion, the isoleucine residue at position 160 in the FKF1 LOV domain plays an important role in the FKF1-mediated flowering regulation through the formation of adequate protein complexes.
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Table of Contents
Abstract ⅰ
Table of Contents ⅲ
List of Figures ⅳ
1. Introduction 1
2. Materials and Methods 10
2.1 Plant materials and Growth conditions 10
2.2 Seeds surface sterilization and stratification 10
2.3 Tobacco transient expression system 10
2.4 Co-Immunoprecipitation analysis 11
2.5 RNA isolation and cDNA construction 12
2.6 Quantitative Real Time PCR (qRT-PCR) 13
2.7 Preparation of Protein Extracts and Western Blot Analysis 14
3. Results 16
3.1 Functional characterization of the FKF1 I160R mutant form in vivo 16
3.1.1 The function of FKF1 I160R protein in the regulation of flowering is enhanced 16
3.1.2 FKF1 I160R protein is less abundant 20
3.1.3 FKF1 I160R protein increases FT mRNA levels without affecting CO transcription 23
3.2 Comparative analysis of the heterodimer formation between FKF1 or FKF1 I160R and known partner proteins in planta or in vivo 26
3.2.1 FKF1 I160R has no significant effect on the binding to GI in vivo 26
3.2.2 A subtle change in the interaction between FKF1 I160R and CO in tobacco leaf tissues 30
3.2.3 FKF1 I160R showed an increase in the protein complex formation with COP1 in vivo, likely leading to CO stabilization 32
4. Discussion 34
5. References 43
6. 국문요약 48
